In our genome-wide screens for small RNAs, we found that a number of short RNAs actually encode small proteins. The correct annotation of the smallest proteins is one of the biggest challenges of genome annotation, and perhaps more importantly, few annotated short ORFs have been confirmed to correspond to synthesized proteins. Although these proteins have largely been missed, the few small proteins that have been studied in detail in bacterial and mammalian cells have been shown to have important functions in signaling and in cellular defenses (1, 2). We thus established a project to identify and characterize proteins of less than 50 amino acids. We used sequence conservation and ribosome binding site models to predict genes encoding small proteins, defined as having 16-50 amino acids, in the intergenic regions of the model E. coli genome. We tested expression of these predicted as well as previously annotated small proteins by integrating the sequential peptide affinity tag directly upstream of the stop codon on the chromosome and assaying for synthesis using immunoblot assays. This approach confirmed that 20 previously annotated and 18 newly discovered proteins of 16-50 amino acids are synthesized. We have now initiated complementary biochemical approaches to identify additional small proteins. Remarkably more than half of the newly discovered proteins are predicted to be single transmembrane proteins. This observation prompted us to examine the localization, topology, and membrane insertion of the small proteins. Biochemical fractionation showed that, consistent with the predicted transmembrane helix, the small proteins generally are most abundant in the inner membrane fraction. Examples of both Nin-Cout and Nout-Cin orientations as well as dual topology were found in assays of topology-reporter fusions to representative small transmembrane proteins. In addition, fractionation analysis of small protein localization in strains depleted of SecE or YidC uncovered differential requirements for these membrane insertion pathways. Thus, despite their diminutive size, small proteins display considerable diversity in topology, biochemical features, and insertion pathways. We now are employing many of the approaches the group has used to characterize the functions of small regulatory RNAs to elucidate the functions of the small proteins. Systematic assays for the accumulation of tagged versions of the proteins have shown that many small proteins accumulate under specific growth conditions or after exposure to stress. We also generated and screened bar-coded null mutants and identified small proteins required for resistance to cell envelope stress and acid shock. In addition, the attached sequential peptide affinity tag is being exploited to identify co-purifying complexes. The combination of these approaches is giving insights into when, where and how the small proteins are acting. For example, we found that synthesis of a 42-amino acid protein, now denoted MntS (formerly the small RNA gene rybA) is repressed by high levels of manganese through MntR. In recent studies we showed that MntS helps manganese activate a variety of enzymes under manganese-poor conditions, while overproduction of MntS leads to very high intracellular manganese and bacteriostasis under manganese-rich conditions (3). These and other phenotypes have led us to propose that MntS modulates intracellular manganese levels, possibly by inhibiting the manganese exporter MntP. We also discovered the 49-amino acid inner membrane protein AcrZ (formerly named YbhT), associates with the AcrAB-TolC multidrug efflux pump, which confers resistance to a wide variety of antibiotics and other compounds. Co-purification of AcrZ with AcrB, in the absence of both AcrA and TolC, two-hybrid assays and suppressor mutations indicate this interaction occurs through the inner membrane protein AcrB. Mutants lacking AcrZ are sensitive to many, but not all, of the antibiotics transported by AcrAB-TolC. This differential antibiotic sensitivity suggests that AcrZ may enhance the ability of the AcrAB-TolC pump to export certain classes of substrates. This work together with our ongoing studies of other small proteins suggest that many are acting as regulators of larger membrane protein complexes.